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CYTOGENETIC CHARACTERIZATION OF THE FLORIDA MANATEE (Trichechus manatus latirostris) BY CHROMOSOME BANDING TECHNIQUES

B Gray1, R Zori1, P McGuire2, S Felton1, R Bonde3

1Division of Genetics, College of Medicine, University of Florida;
2 Department of Biochemistry & Molecular Biology, University of Florida;
3 U.S. Department of the Interior, Florida Integrated Science Center, Florida.

Presented at the 14th Biennial Conference on the Biology of Marine Mammals,
28 November - 3 December 2001, Vancouver, BC, Canada.


ABSTRACT

Published cytogenetic data for the Order Sirenia is limited and, thus far, karyotypes produced have been restricted to conventional or solid chromosome staining techniques. To facilitate identification of individual chromosome homologs for the Florida manatee (Trichechus manatus latirostris), we have applied primary chromosome banding techniques, G- and Q-banding, to metaphase chromosomes prepared from T- and B-cell peripheral blood lymphocyte cell cultures established from six individuals (three males; three females).  Following brightfield and fluorescence microscopic analyses, a previously published modal chromosome number of 48 was confirmed for this species.  Digital imaging methods were subsequently employed and individual homologues were identified by unique G-band patterns and chromosome morphologies.  A standard banded karyotype was constructed, for both sexes, based on the G-band chromosome pattern obtained in these studies.  Characterization of additional cytogenetic features of this species by supplemental chromosome banding techniques, C-banding (constitutive heterochromatin), Ag-NOR staining (nucleolar organizer regions), and DA/DAPI staining was also performed. Cytogenetic features, including chromosome morphology and banding patterns, of Trichechus manatus latirostris are described. These studies may provide a basis for more precise inter and intra specific comparisons by cytogenetic methodologies.


INTRODUCTION

     Of the four species of the modern sirenians (manatees and dugongs), we know of only one, the Amazonian manatee (Trichechus inunguis), which has been studied by cytogenetic methods that included the use of chromosome banding techniques (Assis et al., 1988). Published karyotypic data for the remaining species, has been limited to the Florida manatee (Trichechus manatus latirostris) and the dugong (Dugong dugon). Unfortunately, the existing cytogenetic studies have been restricted to solid stained chromosomes, which prohibit the precise identification of individual chromosome homologs, chromosome regions, and/or chromosome bands.
     A renewed interest in the chromosomal or cytogenetic status of various species has been generated by the advancements of genetic mapping techniques utilizing fluorescence in situ hybridization (FISH) techniques and related comparative genomic techniques such as Zoo-FISH. This type of work would undoubtedly benefit from the availability of a more detailed or precise karyotype for the species being studied.
     We present the first karyotypic data generated by multiple chromosome banding techniques, including G-banding, Q-banding, C-banding, Ag-NOR staining, and DA/DAPI staining for the Florida manatee (Trichechus manatus latirostris).   Combining the chromosome morphology and banding patterns, we were able to identify each homolog pair and consistently construct an accurate karyotype for six individuals.


MATERIALS AND METHODS

Blood Collection & Culture:
The medial aspect of the manatee's flipper was surgically scrubbed and sterilized.  Venipuncture was accomplished through a sterile 18-20 gauge, 1.5 inch needle inserted into the palpable interosseous space between the radius and ulna.  This blind stick includes target vessels within a small plexus (brachial vascular bundle) deep within the tissue.  Vacutainers were used to draw approximately 20 mls of blood into green top sodium heparin tubes.  Samples were placed on ice and transported to the laboratory as soon as possible for cytogenetic processing.  Transit time ranged from 3 to 36 hours.

Buffy coats obtained from the blood were cultured at 36oC for 72 hours in RPMI medium (supplemented with 20% fetal bovine serum, 0.8% L-glutamine, and 0.4% penicillin-strepomycin) with 150l of phytohemagglutinin (L & M-forms) or pokeweed.  Mitotic cells were harvested following modifications of the procedure as described by Brown MG (1997).

Chromosome Preparation:
Microscope slides were cleaned by dipping in methanol followed by a rinse in deionized water.   The slides were covered with distilled water and stored at 4C until ready for use.   The lymphocyte suspension was diluted with fix solution to achieve a slightly cloudy mix.  Several drops of this suspension were placed on a cold and wet microscope slide and allowed to air dry.  Metaphase spreading was assessed by phase microscopy and adjustments were made to accommodate drying times affecting spreading and residual cytoplasm.

Chromosome Banding:
G-banded chromosome preparations were obtained for all six individuals by modifications of the trypsin (GTG) and Giemsa staining procedure as described by Seabright, M (1971).
Q-banding was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Gustashaw, KM (1997).
C-banding was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Benn, PA and MA Perle (1986).
Ag-NOR staining was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Verma, RS and KA Babu(1984).
DA/DAPI staining was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Benn, PA and MA Perle (1986)

Imaging and Karyotype Production:
A minimum of three metaphase images and karyotypes per individual were prepared by computer digital imaging methods.

clear pixel Florida Manatee - click to enlarge clear pixel clear pixel clear pixel clear pixel clear pixel clear pixel clear pixel clear pixel
Figure 1 a-b - click to enlarge

Figures 1a-b:  a: G-banded metaphase cell at 1000x;  b: Q-banded metaphase cell at 1000x

Figure 2 - click to enlarge
Table 1  - click to enlarge

Figure 2:  Preliminary G-banded karyotype (male) arranged by chromosome relative sizes only

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Figure 3a - click to enlarge

Figure 3a: G-banded karyotype (male)  with chromosomes grouped by morphology; st=subtelomeric; sm=submetacentric; m=metacentric

Figure 4 - click to enlarge

Figure 4:  C-banded metaphase and preliminary karyotype arranged by chromosome morphologies

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Figure 3b - click to enlarge

Figure 3b: G-banded karyotype (female) with chromosomes grouped by morphology; st=subtelomeric; sm=submetacentric; m=metacentric

Figure 5 - click to enlarge

Figure 5:  Ag-NOR stained metaphase; arrows indicate positive stained regions

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RESULTS

     As previously published for T. manatus latirostris (White et al., 1976), the 2N number of 48 was present in all individuals examined in this study.  The G-banded metaphase cells demonstrated distinct and unique patterns that, when combined with morphology, permitted the identification of each pair of homologs (Figure1a). The Q-banded pattern was similar to the G-banded pattern obtained (Figure 1b).   Sex chromosome identification was determined based on a presumed X/Y system and the observation of two non-homologous chromosomes in presumed males (three male and three female karyotypes were accurately correlated with the phenotypic gender post karyotyping).
     A preliminary karyotype was constructed based on the G-banding pattern and chromosome relative size (Figure 2). Subsequently, this karyotype was reconfigured to incorporate the chromosome morphology with chromosome relative size and G-banding pattern. The chromosome morphologies (centromeric position) observed in the six specimens studied consisted of three primary subtypes; subterminal (st), median (m), and submedian (sm)*. The resulting karyotype was divided into seven groups (A-G) as described in Table 1 and depicted in Figures 3a-b.
     C-banding positive regions appeared to be restricted to the centromeric regions in all chromosome pairs (Figure 4). C-band positive secondary constrictions were not identified.
     Ag-NOR stained positive regions were identified on two chromosomes of similar morphology (Figure 5) in both individuals studied.
     DA/DAPI staining did not demonstrate any detectable differentially stained regions in the two individuals studied with this technique.

*commonly accepted synonyms: subtelocentric, metacentric, and submetacentric


DISCUSSION

     To our knowledge, the cytogenetic studies presented here represent the first published banded karyotype for the Florida manatee (Trichechus manatus latirostris). The resulting G-banded karyotypes provide a basis for future inter and intra species chromosome comparisons by facilitating the communication and description of specific chromosomes, chromosome regions, and/or chromosome bands.
     At this time, we have chosen to group the chromosomes based on morphology and terminology as described by Levan et al. (1964), avoiding the use of the term acrocentric, which might imply the presence of additional NOR regions yet to be demonstrated. However, we have identified positive Ag-NOR staining regions in the two individuals studied thus far by this method. These NOR regions appear to reside in a single pair of chromosomes, presumed to be homologous and tentatively assigned as chromosome pair 20 (see Figure 3a-b). This is similar to that reported in the Amazonian manatee (T. inunguis) and the morphology and G-banded pattern for these chromosome appears quite similar, suggesting that this chromosome may be well conserved between these two species.  Assis et al. (1988) proposed Robertsonian type rearrangements (chromosome fissions) as the mechanism for karyotypic divergence between these two species and our initial comparison of the banding patterns of the smaller subtelocentric chromosomes in T. inunquis with the banding patterns observed in some of the metacentric chromosomes in T. manatus latirostris are consistent with this proposal.  Further comparisons of the banding patterns are underway.
     We anticipate further refinement of this species' karyotype, as more individuals are studied. Such refinements may include the identification of polymorphic regions (chromosomal variants) and identification of sub-bands.  These efforts will include chromosome measurements (p/q ratios) and construction of a standardized ideogram for future use and comparisons.  Subsequent assignments of genetic mapping data by various techniques such as zoo-FISH, should now prove more accurate with this species.


REFERENCES

Assis, M. F. L., R. C. Best, R. M. S. Barros and Y. Yonenagayassuda. 1988. Cytogenetic Study of Trichechus-Inunguis (Amazonian Manatee). Revista Brasileira De Genetica 11:41-50.

Benn, P. A. and M. A. Perle. 1986. Chromosome staining and banding techniques. 57-84 in D. E. Rooney and B. H. Czepulkowski, ed. Human Cytogenetics: A Practical Approach. IRL Press Co., Oxford.

Brown, M. G. and H. J. Lawce. 1997. Peripheral blood cytogenetic methods. 77-171 in M. J. Barch, T. Knutsen and J. L. Spurbeck, ed. The AGT Cytogenetics Laboratory Manual. Lippincott-Raven Publishers, Philadelphia.

Gustashaw, K. M. 1997. Chromosome stains. 266-268 in M. J. Barch, T. Knutsen and J. L. Spurbeck, ed. The AGT Cytogenetics Laboratory Manual. Lipppincott-Raven Publishers, Philadelphia.

Levan, A., K. Fredga and A. Sandberg. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201-220.

Loughman, W., F. Frye and E. Herald. 1970. The chromosomes of a male manatee. International Zoo Yearbook 10:151-152.

Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet 2:971-972.

Verma, R. S. and K. A. Babu. 1984. Silver staining techniques of nucleolar organizer regions (NORs): Principles and methodology. Karyogram 10:4-5.

White, J. R., D. R. Harkness, R. E. Isaacks and D. A. Duffield. 1976. Some Studies on Blood of Florida Manatee, Trichechus-Manatus Latirostris. Comparative Biochemistry and Physiology a-Physiology 55:413-417.

White, J. R., D. R. Harkness, R. E. Isaacks and D. A. Duffield. 1977. Trichechus manatus latirostris (manatee). Order: Sirenia. Family: Trichechidae. in T. C. Hsu and K. Benirschke, ed. An atlas of mammalian chromosomes. Springer-Verlag, New York, Heidelberg, & Berlin.


ACKNOWLEDGMENTS

We would like to thank Alicia Bethke and Kristen Drown for all of their expert technical assistance in this project. Drs. Dave Murphy, Mark Lowe, Maya Daugherty and staff of the Florida Marine Research Institute provided additional support during sample collection.


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